RESUMO
Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.
Assuntos
Antipsicóticos/uso terapêutico , Autofagia , Mitofagia , Esclerose Múltipla/tratamento farmacológico , Animais , Antipsicóticos/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/sangue , Proteínas Relacionadas à Autofagia/líquido cefalorraquidiano , Axônios/efeitos dos fármacos , Axônios/metabolismo , Biomarcadores/metabolismo , Clozapina/farmacologia , Citocinas/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Glucose/metabolismo , Haloperidol/farmacologia , Inflamação/patologia , Interleucina-1beta/metabolismo , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Modelos Biológicos , Atividade Motora/efeitos dos fármacos , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/fisiopatologia , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Citocinas , Fator 2 de Crescimento de Fibroblastos , Lipopolissacarídeos , Doenças Neuroinflamatórias , Animais , Camundongos , Doenças Neuroinflamatórias/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Citocinas/metabolismo , Células Cultivadas , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Neuroglia/metabolismo , Neuroglia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
In recent years, many pre-clinical studies have tested gene therapy approaches as possible treatments for epilepsy, following the idea that they may provide an alternative to conventional pharmacological and surgical options. Multiple gene therapy approaches have been developed, including those based on anti-sense oligonucleotides, RNA interference, and viral vectors. In this opinion article, we focus on translational issues related to viral vector-mediated gene therapy for epilepsy. Research has advanced dramatically in addressing issues like viral vector optimization, target identification, strategies of gene expression, editing or regulation, and safety. Some of these pre-clinically validated potential gene therapies are now being tested in clinical trials, in patients with genetic or focal forms of drug-resistant epilepsy. Here, we discuss the ongoing translational research and the advancements that are needed and expected in the near future. We then describe the clinical trials in the pipeline and the further challenges that will need to be addressed at the clinical and economic levels. Our optimistic view is that all these issues and challenges can be overcome, and that gene therapy approaches for epilepsy will soon become a clinical reality.
Assuntos
Epilepsia , Terapia Genética , Humanos , Epilepsia/genética , Epilepsia/terapia , Vetores Genéticos/genética , Oligonucleotídeos AntissensoRESUMO
HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.
Assuntos
Encéfalo/patologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/virologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/virologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Produtos do Gene tat/farmacologia , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Transgênicos , Neurotransmissores/metabolismo , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Estatísticas não Paramétricas , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.
Assuntos
Líquidos Corporais , Cauda , Animais , Ratos , Plasma , Coleta de Amostras Sanguíneas , EletroencefalografiaRESUMO
Neuroinflammation represents a dynamic process of defense and protection against the harmful action of infectious agents or other detrimental stimuli in the central nervous system (CNS). However, the uncontrolled regulation of this physiological process is strongly associated with serious dysfunctional neuronal issues linked to the progression of CNS disorders. Moreover, it has been widely demonstrated that neuroinflammation is linked to epilepsy, one of the most prevalent and serious brain disorders worldwide. Indeed, NLRP3, one of the most well-studied inflammasomes, is involved in the generation of epileptic seizures, events that characterize this pathological condition. In this context, several pieces of evidence have shown that the NLRP3 inflammasome plays a central role in the pathophysiology of mesial temporal lobe epilepsy (mTLE). Based on an extensive review of the literature on the role of NLRP3-dependent inflammation in epilepsy, in this review we discuss our current understanding of the connection between NLRP3 inflammasome activation and progressive neurodegeneration in epilepsy. The goal of the review is to cover as many of the various known epilepsy models as possible, providing a broad overview of the current literature. Lastly, we also propose some of the present therapeutic strategies targeting NLRP3, aiming to provide potential insights for future studies.
RESUMO
A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Epilepsia/genética , Epilepsia/terapia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Convulsões/genética , Convulsões/terapia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Proliferação de Células , Epilepsia/metabolismo , Epilepsia/patologia , Fator 2 de Crescimento de Fibroblastos/genética , Terapia Genética , Vetores Genéticos/genética , Masculino , Neurogênese , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismo , Convulsões/patologia , Resultado do TratamentoRESUMO
Temporal lobe epilepsy often manifests months or even years after an initial epileptogenic insult (e.g., stroke, trauma, status epilepticus) and, therefore, may be preventable. However, no such preventive treatment is currently available. Aim of this study was to test an antioxidant agent, 7,8-dihydroxyflavone (7,8-DHF), that is well tolerated and effective in preclinical models of many neurological disorders, as an anti-epileptogenic drug. However, 7,8-DHF also acts as a TrkB receptor agonist and, based on the literature, this effect may imply an anti- or a pro-epileptogenic effect. We found that low- (5 mg/kg), but not high-dose 7,8-DHF (10 mg/kg) can exert strong anti-epileptogenic effects in the lithium-pilocarpine model (i.e., highly significant reduction in the frequency of spontaneous seizures and in the time to first seizure after status epilepticus). The mechanism of these different dose-related effects remains to be elucidated. Nonetheless, considering its excellent safety profile and antioxidant properties, as well as its putative effects on TrkB receptors, 7,8-DHF represents an interesting template for the development of effective and well-tolerated anti-epileptogenic drugs.
Assuntos
Epilepsia , Flavonas , Estado Epiléptico , Animais , Antioxidantes/uso terapêutico , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/prevenção & controle , Receptor trkB , Convulsões , Modelos Animais de DoençasRESUMO
PURPOSE: We have recently reported that viral vector-mediated supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) in a lesioned, epileptogenic rat hippocampus limits neuronal damage, favors neurogenesis, and reduces spontaneous recurrent seizures. To test if this treatment can also prevent hippocampal circuit reorganization, we examined here its effect on mossy fiber sprouting, the best studied form of axonal plasticity in epilepsy. METHODS: A herpes-based vector expressing FGF-2 and BDNF was injected into the rat hippocampus 3 days after an epileptogenic insult (pilocarpine-induced status epilepticus). Continuous video-electroencephalography (EEG) monitoring was initiated 7 days after status epilepticus, and animals were sacrificed at 28 days for analysis of cell loss (measured using NeuN immunofluorescence) and mossy fiber sprouting (measured using dynorphin A immunohistochemistry). KEY FINDINGS: The vector expressing FGF-2 and BDNF decreased both mossy fiber sprouting and the frequency and severity of spontaneous seizures. The effect on sprouting correlated strictly with the cell loss in the terminal fields of physiologic mossy fiber innervation (mossy cells in the dentate gyrus hilus and CA3 pyramidal neurons). SIGNIFICANCE: These data suggest that the supplementation of FGF-2 and BDNF in an epileptogenic hippocampus may prevent epileptogenesis by decreasing neuronal loss and mossy fiber sprouting, that is, reducing some forms of circuit reorganization.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica/genética , Hipocampo/patologia , Fibras Musgosas Hipocampais/patologia , Regeneração Nervosa/genética , Estado Epiléptico/patologia , Animais , Citomegalovirus , Dinorfinas/genética , Eletroencefalografia , Vetores Genéticos , Hipocampo/efeitos dos fármacos , Masculino , Fibras Musgosas Hipocampais/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Ratos , Ratos Sprague-Dawley , Processamento de Sinais Assistido por Computador , Estado Epiléptico/induzido quimicamente , Gravação em VídeoRESUMO
A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.
RESUMO
Epilepsy is a serious neurological disorder affecting about 1% of the population worldwide. Epilepsy may arise as a result of acquired brain injury, or as a consequence of genetic predisposition. To date, genome-wide association studies and exome sequencing approaches have provided limited insights into the mechanisms of acquired brain injury. We have previously reported a pro-epileptic gene network, which is conserved across species, encoding inflammatory processes and positively regulated by sestrin3 (SESN3). In this study, we investigated the phenotype of SESN3 knock-out rats in terms of susceptibility to seizures and observed a significant delay in status epilepticus onset in SESN3 knock-out compared to control rats. This finding confirms previous in vitro and in vivo evidence indicating that SESN3 may favour occurrence and/or severity of seizures. We also analysed the phenotype of SESN3 knock-out rats for common comorbidities of epilepsy, i.e., anxiety, depression and cognitive impairment. SESN3 knock-out rats proved less anxious compared to control rats in a selection of behavioural tests. Taken together, the present results suggest that SESN3 may regulate mechanisms involved in the pathogenesis of epilepsy and its comorbidities.
RESUMO
Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1ß expression. The effect appeared to be most prominent on IL-1ß, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1ß in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hipocampo , Inflamação/patologia , Convulsões/patologia , Convulsões/prevenção & controle , Animais , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Interleucina-1beta/metabolismo , Distribuição Aleatória , Ratos , Recidiva , Convulsões/metabolismo , Convulsões/fisiopatologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologiaRESUMO
Expression and release of nociceptin/orphanin FQ (N/OFQ) are elevated in the substantia nigra reticulata of 6-hydroxydopamine-hemilesioned rats, suggesting a pathogenic role for N/OFQ in Parkinson's disease. In this study, we investigated whether elevation of N/OFQ expression in 6-hydroxydopamine-hemilesioned rats selectively occurs in substantia nigra and whether hypomotility following acute haloperidol administration is accompanied by a rise in nigral N/OFQ levels. Moreover, to prove a link between N/OFQ and idiopathic Parkinson's disease in humans, we measured N/OFQ levels in the cerebrospinal fluid of parkinsonian patients undergoing surgery for deep brain stimulation. In situ hybridization demonstrated that dopamine depletion was associated with increase of N/OFQ expression in substantia nigra (compacta +160%, reticulata +105%) and subthalamic nucleus (+45%), as well as reduction in caudate putamen (-20%). No change was observed in globus pallidus, nucleus accumbens, thalamus, and motor cortex. Microdialysis coupled to the bar test allowed to demonstrate that acute administration of haloperidol (0.8 and 3 mg/kg) increased nigral N/OFQ levels (maximally of +47% and +53%, respectively) in parallel with akinesia. A correlation with preclinical studies was found by analyzing N/OFQ levels in humans. Indeed, N/OFQ levels were found to be approximately 3.5-fold elevated in the cerebrospinal fluid of parkinsonian patients (148 fmol/ml) compared with nonparkinsonian neurologic controls (41 fmol/ml). These data represent the first clinical evidence linking N/OFQ to idiopathic Parkinson's disease in humans. They strengthen the pathogenic role of N/OFQ in the modulation of parkinsonism across species and provide a rationale for developing N/OFQ receptor antagonists as antiparkinsonian drugs.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos Opioides/metabolismo , Doença de Parkinson/patologia , Adrenérgicos/toxicidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Animais , Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Haloperidol/uso terapêutico , Humanos , Masculino , Microdiálise/métodos , Pessoa de Meia-Idade , Peptídeos Opioides/líquido cefalorraquidiano , Peptídeos Opioides/genética , Oxidopamina/toxicidade , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologia , Ratos , Ratos Sprague-Dawley , Adulto Jovem , NociceptinaRESUMO
Neurotrophic factors are involved in the survival of neurons as well as in the proliferation and differentiation of neuronal precursors. Therefore, modulating their levels in lesion areas may exert favorable effects on seizure-induced damage. However, it is unclear if damage limitation or repair may prevent epileptogenesis; it is also uncertain which neurotrophic factor should be administered for limiting or repairing damage while avoiding possible proepileptic effects. We used viral vectors to locally supplement fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when an epileptogenic damage was already in place. These vectors were tested in the pilocarpine model of status epilepticus-induced neurodegeneration and epileptogenesis. FGF-2/BDNF expressing vectors increased neuronogenesis, limited neuronal damage, and reduced the occurrence of spontaneous seizures. These findings are discussed with consideration of the hurdles that will have to be overcome before clinical application.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia Pós-Traumática/tratamento farmacológico , Fatores de Crescimento Neural/uso terapêutico , Anticonvulsivantes/farmacologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Epilepsia Pós-Traumática/prevenção & controle , Epilepsia Pós-Traumática/terapia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Terapia Genética , Vetores Genéticos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Humanos , Fatores de Crescimento Neural/farmacologiaRESUMO
Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.
Assuntos
Epilepsia/genética , Fator 2 de Crescimento de Fibroblastos/genética , Predisposição Genética para Doença/genética , Degeneração Neural/genética , Plasticidade Neuronal/genética , Animais , Morte Celular/genética , Convulsivantes/farmacologia , Modelos Animais de Doenças , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Feminino , Ácido Glutâmico/metabolismo , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Células Piramidais/citologia , Células Piramidais/metabolismo , Células Piramidais/fisiopatologia , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestruturaRESUMO
Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)(5)]NPS was identified. [D-Cys(tBu)(5)]NPS up to 100 microM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pK(B), 6.62). In Schild analysis experiments [D-Cys(tBu)(5)]NPS (0.1-100 microM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 6.44. Ten micromolar [D-Cys(tBu)(5)]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)(5)]NPS (1-10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)(5)]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.
Assuntos
Neuropeptídeos/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Peptídeos/farmacologiaRESUMO
Gene therapy has recently advanced to the level of standard of care for several diseases. However, its application to neurological disorders is still in the experimental phase. In this review, we discuss recent advancements in the field that provide optimism on the possibility to have first-in-human studies for gene therapy of some forms of epilepsy in the not so distant future.
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Neurological disorders affecting the central nervous system (CNS) are still incompletely understood. Many of these disorders lack a cure and are seeking more specific and effective treatments. In fact, in spite of advancements in knowledge of the CNS function, the treatment of neurological disorders with modern medical and surgical approaches remains difficult for many reasons, such as the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ due to the blood-brain barrier. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. Gene therapy can result in a stable or inducible expression of transgene(s), and can allow a nearly specific expression in target cells. In this review, we will discuss the most commonly used tools for the delivery of genetic material in the CNS, including viral and non-viral vectors; their main applications; their advantages and disadvantages. We will discuss mechanisms of genetic regulation through cell-specific and inducible promoters, which allow to express gene products only in specific cells and to control their transcriptional activation. In addition, we will describe the applications to CNS diseases of post-transcriptional regulation systems (RNA interference); of systems allowing spatial or temporal control of expression [optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)]; and of gene editing technologies (CRISPR/Cas9, Zinc finger proteins). Particular attention will be reserved to viral vectors derived from herpes simplex type 1, a potential tool for the delivery and expression of multiple transgene cassettes simultaneously.
RESUMO
RATIONALE: Nociceptin/orphanin FQ (N/OFQ) has been proposed to be a functional antagonist of corticotropin-releasing factor (CRF) in relation to its anti-stress action and its ability to antagonize the anorectic effect of CRF in rats without exhibiting affinity for CRF receptors. The bed nucleus of the stria terminalis (BST) is highly sensitive to the inhibitory effect of N/OFQ on CRF-induced anorexia. OBJECTIVE: The present study was aimed at further evaluating the role of the BST in the functional antagonism between N/OFQ and CRF by examining it at molecular level and in the context of CRF-induced anxiety in the rat. MATERIALS AND METHODS: First, in situ hybridization experiments investigated the expression of the pro-N/OFQ precursor and of NOP receptors in several brain areas 6 h after injection of CRF (0.2 and 1 microg/rat) into the lateral cerebroventricle (LV). Second, the elevated plus maze test was used to evaluate whether N/OFQ, injected into the BST (0.05 and 0.5 microg/rat) or into the LV (0.5, 1.8, and 2.4 microg/rat), inhibits the anxiogenic-like effect evoked by LV injection of CRF (1 microg/rat) in rats. RESULTS: The in situ hybridization study showed that LV injection of CRF 1 microg/rat increases NOP receptor expression in the BST, while no change of the N/OFQ precursor was observed. On the other hand, N/OFQ injection into the BST blocks the anxiogenic effect of CRF at doses lower than those required by LV injection (0.5 vs 1.8 microg/rat, respectively). CONCLUSION: These data provide further support for the hypothesis that N/OFQ may behave as functional antagonist of CRF and suggest that this antagonism may occur within the BST.
Assuntos
Hormônio Liberador da Corticotropina/fisiologia , Peptídeos Opioides/fisiologia , Núcleos Septais/fisiologia , Animais , Ansiedade/induzido quimicamente , Ansiedade/tratamento farmacológico , Hormônio Liberador da Corticotropina/farmacologia , Hibridização In Situ , Injeções Intraventriculares , Masculino , Aprendizagem em Labirinto , Peptídeos Opioides/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptores Opioides/biossíntese , Núcleos Septais/metabolismo , Receptor de Nociceptina , NociceptinaRESUMO
Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.